Porphyromonas gingivalis proteinase and activator of keratinoncyte collagenolysis: Purification, charactization and cloning The finding that a post-log phase, cell-free media of a Porphyromonas gingivalis (P.g.) culture can stimulate collagenolytic activity in a keratinocyte culture system has led to work led to an investigative of the factor(s) in the P.g. culture media responsible for this activity. Through various purification schemes, it was found that the P.g. culture supernatant contained various molecular weight species whose relative abundance were concentration dependant. Biochemical analysis of the numerous proteolytic species in P.g. culture supernatant suggested that they are high molecular weight aggregates (>52 kDa) of lower molecular weight species (>=52 kDa) which associate in a non-covalent manner. N-ternimal amino acid sequencing and monoclonal antibody Western blot analysis of the low monoclonal weight peptides confirmed that the proteolytic species are related through their cross- immunoreactivity. This work suggests that thiol-protease isoforms exist in Porphyromonas gingivalis culture. Immunoprecipitation of 35s L-cysteine labeled P.g. cell extracts from early log-phase culture and of pulse labeled extra-cellular culture supernatant precipitated a 48 kDa species which disappeared as a 100 kDa species arose during the culture period suggesting the that 100 kDa proteinase species is a dimer. The proteinase was found to have hemagglutinin activity consistent with a partial homology to the deduced amino acid sequence of a recently cloned hemagglutinin from P.G.. Work is underway to clone the proteinase by probing the genome and RNA with degenerate oligonucleotide probes which correspond to the N-terminal amino acid sequences of the related peptides. Ph.D. in Biochemistry with clinical specialization in Periodontics.